With regard to a conventional effective utilization of unused resources, particularly biomass (e.g., wood waste material and the like), a major goal was to hydrolyze it into glucose and to convert the glucose into an alcohol. However, even if it was converted into an alcohol, since its added value is low, it was unreasonable to put it into practical use. In addition, by using a polysaccharide (which exists unlimitedly in unused resources) as a raw material, when it is incompletely degraded, an oligosaccharide can be produced. Its application is also being developed as a value-added product having a functionality.
The present inventors started focusing on the physiological activity of rare sugars and confirming it by experiments using cells. 21 century is also called a century of life science, and at present, a DNA study and a protein study are being advanced internationally. Speaking of sugar in post-genomic study, the study is centered on sugar chains, however, in Kagawa Medical University (at present, the Faculty of Medicine in Kagawa University) and the Faculty of Agriculture in Kagawa University to which the present inventors belong, monosaccharides are focused on and their application study such as whether or not the monosaccharides have a physiological activity has been advanced. The background includes the fact that in the Faculty of Agriculture in Kagawa University, a long series of exhaustive studies on the production of rare sugars has been continued for a long time, and recently, a technique of the mass production of part of rare sugars was established. In also Kagawa Medical University (at present, the Faculty of Medicine in Kagawa University), a study of searching for a physiological activity in sugars was started several years ago. A study of searching for a physiological activity by using rare sugars (monosaccharides) produced in the Faculty of Agriculture in Kagawa University was started from 1999 as a regional leading study in a manner that both studies were put together, and it was found that they have various physiological activities.
Monosaccharides are classified broadly into aldoses (sugars having an aldehyde group as a carbonyl group), ketoses (sugars having a ketone group as a carbonyl group) and sugar alcohols (another name: polyols, sugars without a carbonyl group) based on the state of a reducing group (carbonyl group). There is a monosaccharide called “rare sugar”. A rare sugar is defined as a sugar that rarely exists in nature according to the definition of International Society of Rare Sugars. There are many types of rare sugars whose yield is small by an organic chemical synthetic method depending on the type. Accordingly, the present situation is that there are also many types of rare sugars whose property is not known, and that there are many unknown properties for aldohexose (aldose) rare sugars including allose.
In application studies of sugars, conventionally, with regard to the correlation between a sugar and cancer, for example, as described in Patent Document 1, a polysaccharide that is effective in prevention of cancer is known. In addition, there are reports that an oligosaccharide has an effect on counteracting constipation by utilizing the fact that it has an action of regulating the intestines thereby to cause less colon cancer, and recently that a polysaccharide such as agaricus has an effect on suppressing cancer, and also a report on the correlation between a sugar chain and cancer metastasis. Further, in Patent Document 2, an anti-tumor agent containing a derivative of D-allose as the active ingredient is disclosed. On the other hand, as for utilizing a property of a saccharide against active oxygen, for example, as described in Patent Document 3, an inhibitor of active oxygen production containing a polysaccharide having a property of inhibiting active oxygen is known.
Among monosaccharides, psicose is a hexose having a ketone group as a reducing group. It is known that psicose exists as optical isomers in D-form and L-form. Here, although D-psicose is a known substance, it rarely exists in nature, therefore, it is defined as a “rare sugar” according to the definition of International Society of Rare Sugars. However, it became relatively easy to obtain this D-psicose recently, though it is expensive, because of the emergence of epimerase (e.g., see Patent Document 4). According to this official gazette, it is suggested that the prepared D-psicose can be effectively utilized as a sweetener, a carbon source for fermentation, a reagent, a raw material or an intermediate for a cosmetic or a medical product or the like. According to this official gazette, as the sweetener, it only describes a direction of application that it can be utilized in improving the taste for sweetening an oral intake item such as food and drink, feed, toothpaste or a medicine for internal use. With regard to L-psicose that is an optical isomer of D-psicose, the fact that it can be utilized as an edible composition is disclosed in, for example, Patent Document 5 in detail.
On the other hand, an example of application of psicose as an intermediate material for a reagent, a medical product or the like is shown below. For example, according to Non-Patent Document 1, an example of synthesis of a hydantoin derivative using D-psicose as a raw material is reported. In addition, according to Non-Patent Document 2, an example of synthesis of D-fructofranosyl nucleoside is disclosed. In any prior art, it is only reported that D-psicose can be utilized as a raw material or an intermediate for a medical product or the like.
In addition, in Patent Document 6, it is only described that a kojic acid glycoside having a hexose in its structure is excellent in an action of inhibiting melanogenesis, high in stability, high in solubility in water and suitable as the active ingredient of a whitening preparation for external use. In addition, in Patent Document 7, it is described that psicose promotes recovery of the skin barrier function and is useful in preventing such as abnormality of the epidermal growth due to a decrease in the epidermal function of the skin, and the usefulness as a humectant is only described. In addition, a healthy diet for prevention of a disease accompanied by high blood glucose and prevention of obesity containing several saccharides including D-tagatose as an active ingredient is disclosed in Patent Document 8, however, the function of the rare sugar itself is not described. In addition, in Patent Document 9, the application, as an antihyperlipidemic agent, of a polysaccharides complex containing, as the major constituent sugars, arabinose, ribose and glucose including D-sorbose which is one of ketohexoses is only published.
However, in order to advance the study of application of a rare sugar by focusing on a “monosaccharide”, and moreover, in the case where a new application has been completed, it is necessary to establish a technique of mass production of a rare sugar.
On the other hand, L-rhamnose isomerase produced by Pseudomonas stutzerii LL172 is a known enzyme having the following physicochemical properties published in Non-Patent Document 3.
(i) Action
It is an enzyme catalyzing an isomerization reaction from L-rhamnose to L-rhamnulose and isomerization from L-rhamnulose to L-rhamnose. It is known that it also acts on isomerization between D-allose and D-psicose (Non-Patent Document 3), and it is an enzyme that can produce D-allose from D-psicose. Isomerases are named based on a substrate exhibiting the highest activity, therefore, an enzyme named the same L-rhamnose isomerase were isolated from E. coli and Bacillus subtilis, and the gene sequence encoding it has been reported.
(ii) Substrate Specificity
L-rhamnose and L-rhamnulose are used as a substrate. Other than these, L-lyxose and L-xylulose, L-mannose and L-fructose, D-ribose and D-ribulose, D-allose and D-psicose are used as a substrate.
(iii) Active pH and Optimal pH
The active pH ranges from 7.0 to 10.0 and the optimal pH is 9.0.
(iv) pH Stability
It is stable within the pH range of 6.0 to 11.0 in the case where it is kept at 4° C. for 1 hour at various pH values.
(v) Active Temperature and Optimal Temperature
The active temperature ranges from 40 to 65° C. and the optimal temperature is 60° C.
(vi) Temperature Stability
It is stable at 40° C. for 10 minutes and remains at 90% or more even at 50° C. for 10 minutes.
(vii) Effect of a Chelating Agent
Its activity is hardly inhibited even if it coexists with EDTA or EGTA, which is a chelating agent, during the measurement of its activity.
(viii) Effect of a Metal Ion
About 30% of the activity is inhibited by 1 mM cobalt ion.
(ix) Molecular Weight by the SDS-PAGE Method
It is about 43,000.    Patent Document 1: JP-A-5-112455    Patent Document 2: JP-B-59-40400    Patent Document 3: JP-A-07-285871    Patent Document 4: JP-A-6-125776    Patent Document 5: JP-A-57-129671    Patent Document 6: JP-A-4-198115    Patent Document 7: JP-A-2000-103728    Patent Document 8: JP-A-6-65080    Patent Document 9: JP-A-2-286620    Non-Patent Document 1: Tetrahedron Vol. 47, No. 12/13, pp.2133 (1991)    Non-Patent Document 2: Acta. Chem. Scand. Ser. B. Vol. 38, No. 5, pp. 367 (1984)    Non-Patent Document 3: “Journal of Fermentation and Bioengineering”, Vol. 85, pp. 539 to 541 (1998)